Chemical changes of cellular proteins, also called posttranslational modifications, reflect important and complex biochemical processes directly related to cell biological conditions. Hence, these posttranslational modifications are linked to multiple disease states including cancer and their structural determination represents a prerequisite for definition and development of new targeted therapies. Protein phosphorylation represents one of the most important signaling modifications mandatory for regulation of most of cellular processes. Standard bottom-up proteomic approaches (Aebersold, 2001) based on high performance liquid chromatography/tandem mass spectrometry cannot easily discriminate between non-phosphorylated and phosphorylated peptides due to the effect of negatively-charged phospho-group deteriorating the signal in positive ion electrospray mass spectra, so fractionation and further identification steps are needed. There have been reported chemical and affinity approaches being used for discrimination of phosphopeptides from regular peptides (Bodenmiller, 2007). Chemical methods were based in beta-elimination of a phosphate group. The double bond formed this way then reacted with various nucleophilic agents carrying some functional group which subsequently was used for selective isolation. Phosphoramidate chemistry served as one important example (Zhou, 2001). Affinity techniques have been based on phosphopeptide separation from a complex mixture by affinity interaction with solid substrate. Immobilized metal affinity chromatography (IMAC) or the application of metal oxides, e.g. TiO2, ZrO2, Al2O3 have been usually combined either on-line or off-line with some chromatographic separation. Electrospray (ESI) and/or matrix-assisted laser desorption ionization (MALDI) were common ionization techniques. Most challenging tasks remained the lack of automation and problems associated with small sample volume operations. Therefore, we started with phosphopeptide enrichment directly on MALDI target surface on which the desorption and ionization processes take place. In these experiments the surface, at which the phosphopeptides are going to be analyzed, is modified by application of affinity substrate, which more readily binds phosphopeptides compared to non-phosphorylated compounds. Upon selective elution of the non-phosphorylated material, phosphorylated peptides are determined by standard MALDI mass spectrometry experiment. This approach is easier compared to electrospray ionization (both in on- and offline setup) and lower detection limit can be achieved. The problem remains the discovery of a suitable way of surface modification of a MALDI target. Most common modification materials are titanium and zirconium oxides. The active surface has been prepared in multiple ways mostly suffering from complications both in sample preparation and phosphopeptide analysis. Blacken (Blacken 2007, 2009) employed a reactive landing procedure, which was ultra-low pressure experiment based on surface bombardment by desolvated ions or their clusters prepared from 4B atom propoxides (Løver, 1997). The charged clusters of stable isotopes prepared this way had to be accelerated by external electric field (up to 15 kV) to hypertermal energies being thousand times higher than normal internal laboratory temperature (Blacken 2007, 2009). Kinetic energy of these projectiles was converted into internal energy during the surface-induced collision process and surface modification. Although this technique gave quality and mechanically stable surfaces, the process was long lasting (several hours) and evacuated device was costly. Upon the introduction of a surface into the instrument, high vacuum had to be reached first and then a 3-hour propoxide deposition could start. The process was not high-throughput and price of surface modification carried this way was high. The second reported process was based on TiO2-assisted pulsed laser deposition (Torta, 2009). This latter approach provided low-quality surfaces and was also costly. Standard “wet” electrospray deposition of zirconium oxide on a heated surface represented another alternative. Elevated surface temperature, however, caused the fragile and chapped layer formation (Blacken 2009 and our observations). Titanium oxide also could be directly deposited (Niklew, 2010). This procedure was very difficult as TiO2 is water insoluble and in fact a suspension had to be directly sprayed onto a surface causing clogging the spray needle and ion current instability. Other technical variants are cited in FIGS. 5 and 6 and declare that our approach reported in this application gives the best results ever reported.